低渗介导抽提牛精子线粒体DNA

Hyposmotic Mediated Extraction of Bovine Sperm Mitochondria DNA

目的:建立一种通过低渗介导的简便、有效的抽提冷冻牛精子线粒体DNA(mtDNA)的方法。方法:低渗处理牛精子,结合去污剂破膜释放精子线粒体,饱和酚去蛋白,无水乙醇沉淀DNA,设计线粒体特异性引物进行PCR分析以及通过NlaⅢ酶切对抽提获得的mtDNA作酶切鉴定,并以水解法作对照。结果:低渗法获得的mtDNA的PCR产物电泳分析和酶切鉴定都清楚地显示与预期相符的目的条带,而对照组水解法抽提冷冻牛精子mtDNA存在稳定性差及重复性差等缺点。结论:低渗介导是一种抽提精子mtDNA的有效方法。

Objective： To establish a simple and effective method for extracting bovine sperm mitochondria DNA （mtDNA） in frozen straw. Methods： Hyposmotic treatment, combined with decontaminant, was used to break plasma membrane and release sperm mitochondria. Proteins were removed with pheno, followed by precipita- tion of DNA with ethanol. Specific PCR primers were designed to amplify mtDNA fragment as well as restriction mapping of the mtDNA was analyzed by Nla III digestion. Hydrolsis method was acted as the control. Results： The electrophoresis of the mtDNA PCR products and Nla III restriction mapping showed the expectant target bands by hyposmotic method, while instability and poor reproducibility were showed by hydrolisis method. Conclusion： Hyposmotic mediation would be an effective method for extracting sperm mitochondria DNA.